Our long-term goal is to determine how cells establish and maintain progressive motility to repopulate tissues in response to external signals. In wound repair, soluble growth factors direct fibroblasts and endothelial cells repopulate the immature matrix to form both the supporting matrix and vasculature required to regenerate the tissue structures. The initial migration is driven by stimulatory cues arising from within the wound bed. However, once within the wound bed, the cells must distribute often in the absence of stimuli gradients. A central question is therefore how cells establish the sustained asymmetry required for progressive migration. Cell migration requires asymmetry of biophysical force-related processes;in eukaryotic cells this is likely governed by intracellular signals. At the front, the cells must extend lamellipodia and form new adhesions to stabilize the dominant protrusion, while rear de-adhesion and retraction is required to enable progressive movement. Between these two cell regions, contractility occurs to bring the cell body forward. To productively choreograph these processes, a cell must establish persistent directionality. During the initial grant period we have found that during motility induced by near ubiquitous chemokinetic EGFR ligands, that the initial actviation of PLCy (phospholipase-Cy) establishes an asymmetry of an important membrane moiety, PIP2 (phosphoinositide bisphosphate). Our preliminary data suggest that the biochemical cascades leading to the biophysical processes are regulated at least in part by this membrane moiety and docking site. Thus, we hypothesize that the localized activation of key biochemical signaling cascades required for productive cell motility results from the integration of phospho-inositide asymmetry in the plasma membrane. We propose to test the following postulates: /. That m-calpain (CAPN2), required for rear release during motility, is localized to an activatable peri-plasma membrane locale by binding to PIP2. II. That PKCS-mediated contractility, required to pull the cell body and tail forward, is localized to regions of PLCy activity linked by phospho-inositide turnover. III. That PI3 kinase designates and stabilizes the leading, dominant lamellipod to provide directionality. We focus on human fibroblasts and endothelial cells, with extension to mesenchymal stem cells. These studies will define molecular bases for spatial restriction of receptor signaling and resultant biophysical responses, offering promise for design of 'smart'scaffolds for tissue engineering to support tissue function.